An miRNA derived from amelogenin exon4 regulates expression of transcription factor Runx2 by directly targeting upstream activators Nfia and Prkch.

Amel, the gene encoding the amelogenin protein involved in enamel formation, is highly alternatively spliced. When exon4 is excised, it can form a mature miRNA (miR-exon4) that has previously been suggested to indirectly regulate expression of the Runt-related transcription factor 2 (Runx2) involved in bone development in ameloblasts and osteoblasts. However, the precise mechanism of this regulation is unclear. In this study, we aimed to identify direct targets of miR-exon4. The transcription factor family nuclear factor I/A (NFI/A) is known to negatively regulate expression of Runx2 and is among the most highly predicted direct targets of miR-exon4 that link to Runx2. Immunostaining detected NFI/A in osteoblasts and ameloblasts in vivo, and reporter assays confirmed direct interaction of the Nfia 3'-UTR and miR-exon4. In addition, silencing of Nfia in MC3T3-E1-M14 osteoblasts resulted in subsequent downregulation of Runx2. In a monoclonal subclone (mi2) of MC3T3-E1 cells wherein mature miR-exon4 was functionally inhibited, we observed significantly downregulated Runx2 expression. We showed that NFI/A was significantly upregulated in mi2 cells at both mRNA and protein levels. Furthermore, quantitative proteomics and pathway analysis of gene expression in mi2 cells suggested that miR-exon4 could directly target Prkch (protein kinase C-eta), possibly leading to RUNX2 regulation through mechanistic target of rapamycin kinase activation. Reporter assays also confirmed the direct interaction of miR-exon4 and the 3'-UTR of Prkch, and Western blot analysis confirmed significantly upregulated mechanistic target of rapamycin kinase phosphorylation in mi2 cells. Taken together, we conclude that Nfia and Prkch expression negatively correlates with miR-exon4-mediated Runx2 regulation in vivo and in vitro, suggesting miR-exon4 directly targets Nfia and Prkch to regulate Runx2.

WDR72 regulates vesicle trafficking in ameloblasts.

As the hardest tissue in the human body, tooth enamel formation is a highly regulated process involving several stages of differentiation and key regulatory genes. One such gene, tryptophan-aspartate repeat domain 72 (WDR72), has been found to cause a tooth enamel defect when deleted or mutated, resulting in a condition called amelogenesis imperfecta. Unlike the canonical genes regulating tooth development, WDR72 remains intracellularly and is not secreted to the enamel matrix space to regulate mineralization, and is found in other major organs of the body, namely the kidney, brain, liver, and heart. To date, a link between intracellular vesicle transport and enamel mineralization has been suggested, however identification of the mechanistic regulators has yet to be elucidated, in part due to the limitations associated with studying highly differentiated ameloblast cells. Here we show compelling evidence that WDR72 regulates endocytosis of proteins, both in vivo and in a novel in vitro ameloblast cell line. We elucidate WDR72's function to be independent of intracellular vesicle acidification while still leading to defective enamel matrix pH extracellularly. We identify a vesicle function associated with microtubule assembly and propose that WDR72 directs microtubule assembly necessary for membrane mobilization and subsequent vesicle transport. Understanding WDR72 function provides a mechanistic basis for determining physiologic and pathologic tissue mineralization.

Effects of Early Life Adversity on Tooth Enamel Formation.

In a systemic effort to survive environmental stress, organ systems fluctuate and adapt to overcome external pressures. The evolutionary drive back toward homeostasis makes it difficult to determine if an organism experienced a toxic exposure to stress, especially in early prenatal and neonatal periods of development. Previous studies indicate that primary human teeth may provide historical records of experiences related to stressors during that early time window. To assess the molecular effects of early life adversity on enamel formation, we used a limited bedding and nesting (LBN) mouse model of early life adversity (ELA) to assess changes in the enamel organ gene expression and enamel matrix mineralization. On average, postnatal day 12 (P12) ELA mice weighed significantly less than the controls. When adjusted for animal weight, ELA molar enamel volume was reduced as compared with the controls, and the relative mineral density of molar enamel was significantly increased. There were no obvious changes in enamel matrix crystal morphology or structure in ELA as compared with the control mouse enamel. RNAseq showed extracellular matrix organization to be the most significantly affected GO and reactome pathways, whereas butanote metabolism was the most significantly altered KEGG pathway. Transcripts expressing the enamel matrix proteins amelogenin (Amelx) and enamelin (Enam) were among the top 4 most differentially expressed genes. When evaluating molecular mechanisms for the changes in gene expression in ELA enamel organs, we found significantly increased expression of Dlx3, while transcripts for clock genes Per1 and Nrd1 were downregulated. These findings support the possibility that the developing enamel organ is sensitive to the pressures of early life adversity and produces molecular and structural biomarkers reflecting these challenges.